Keratinocyte growth factor gene therapy ameliorates ulcerative colitis in rats

AIM:To investigate the effect of keratinocyte growth factor(KGF) gene therapy in acetic acid-induced ulcerative colitis in rat model.METHODS:The colitis of Sprague-Dawley rats was induced by intrarectal infusion of 1 mL 5%(v/v) acetic acid.Twenty-four hours after exposed to acetic acid,rats were divided into three experimental groups:control group,attenuated Salmonella typhimurium Ty21a strain(SP) group and SP strain carrying human KGF gene(SPK) group,and they were separately administered orally with 10% NaHCO3,SP or SPK.Animals were sacrificed and colonic tissues were harvested respectively on day 3,5,7 and 10 after administration.Weights of rats,colonic weight/length ratio and stool score were evaluated.Histological changes of colonic tissues were examined by hematoxylin and eosin(HE) staining method.The expression of KGF,KGF receptor(KGFR) and TNF-α were measured either by enzyme-linked immunosorbent assay or Western blotting.Immunohistochemistry was used to detect the cellular localization of KGFR and Ki67.In addition,superoxide dismutase(SOD) activity and malondialdehyde(MDA) contents in the homogenate were measured.RESULTS:Body weight and colonic weight/length ratio were declined in SPK group compared with SP and control groups(body weight:272.78 ± 17.92 g vs 243.72 ± 14.02 g and 240.68 ± 12.63 g,P < 0.01;colonic weight/length ratio:115.76 ± 7.47 vs 150.32 ± 5.99 and 153.67 ± 5.50 mg/cm,P < 0.01).Moreover,pathological changes of damaged colon were improved in SPK group as well.After administration of SPK strain,KGF expression increased markedly from the 3rd d,and remained at a high level till the 10th d.Furthermore,KGFR expression and Ki67 expression elevated,whereas TNF-α expression was inhibited in SPK group.In the group administered with SPK,SOD activity increased significantly(d 5:26.18 ± 5.84 vs 18.12 ± 3.30 and 18.79 ± 4.74 U/mg,P < 0.01;d 7:35.48 ± 3.35 vs 22.57 ± 3.44 and 21.69 ± 3.94 U/mg,P < 0.01;d 10:46.10 ± 6.23 vs 25.35 ± 4.76 and 27.82 ± 6.42 U/mg,P < 0.01) and MDA contents decreased accordingly(d 7:7.40 ± 0.88 vs 9.81 ± 1.21 and 10.45 ± 1.40 nmol/mg,P < 0.01;d 10:4.36 ± 0.62 vs 8.41 ± 0.92 and 8.71 ± 1.27 nmol/mg,P < 0.01),compared with SP and control groups.CONCLUSION:KGF gene therapy mediated by attenuated Salmonella ameliorates ulcerative colitis induced by acetic acids,and it may be a safe and effective treatment for ulcerative colitis.

Issues within Keratinocyte Growth Factor （KGF） research

As the seventh member of Fibroblast Growth Factor （FGF） family, Keratinocyte Growth Factor （KGF or FGF-7） is observed tp mediate epithelial cell proliferation and differentiation in a variety of tissues. In this article, such following issues within KGF research were reviewed, as （1） KGF functioning pathways： experimental results demonstrated the paracrine pathway of KGF played main role in mesen- chymal-epithelial interactions whereas KGF itself was under the control of a feedback regulation, autocrine provided KGF alternative functioning way particularly in tumourogenesis; （2） KGF in apoptosis： a few of investigations recently illustrated KGF mediated cell survival was based on its mitogenic function via stimulating cell growth, moreover KGF could inhibit the ROS-induced apoptosis through Nrf-2 pathway; （3） KGF during tumourogenesis： high expression of KGF enhanced progression, motility and invasiveness of tumor cells and various cancers, in company with paracrine loop replaced by autocrine loop, meanwhile KGF clearly played the early signal in the progression of breast cancer; （4） Medical application and administration of KGF： KGF had been successfully used in several preclinical models of radiation and chemotherapy-induced mucositis, and developed into commercial medicine （i.e. Palifermin ）, however more effective delivery systems are still under trial.

Keratinocyte growth factor phage model peptides can promote epidermal cell proliferation without tumorigenic effect

Background Keratinocyte growth factor （KGF） significantly influences epithelial wound healing. The aim of this study was to isolate KGF phage model peptides from a phage display 7-mer peptide library to evaluate their effect on promoting epidermal cell proliferation. Methods A phage display 7-mer peptide library was screened using monoclonal anti-human KGF antibody as the target. Enzyme linked immunosorbent assay （ELISA） was performed to select monoclonal phages with good binding activity. DNA sequencing was done to find the similarities of model peptides. Three-（4,5-dimethylthiazol-2-yl） -2,5-diphenyl tetrazolium bromide （MTT） assay, immunofluorescence assay and quantitative real-time PCR analysis were employed to evaluate the effect of the phage model peptides on epidermal cells. Results Thirty-three out of fifty-eight （56.9%） of the isolated monoclonal phages exhibited high binding activity by ELISA. Ten of fifteen obtained phage model peptides were similar to KGF or epidermal growth factor （EGF）. MTT assay data showed that four （No. 1-4） of the ten phage model peptides could promote epidermal cell proliferation. The expression of keratinocyte growth factor receptor （KGFR） mRNA in the KGF control group and the two phage model peptide groups （No. 1 and No. 2） increased. Expression of c-Fos mRNA and c-Jun mRNA in the KGF control group increased, but did not increase in the four phage model peptide groups （No.1-4）. Conclusion Four phage model peptides isolated from the phage display 7-mer peptide library can safely promote epidermal cell proliferation without tumorigenic effect.

Impaired upregulation of keratinocyte growth factor in injured lungs induced by Pseudomonas aeruginosa in immunosuppressed rats

Background The number of immunosupressed patients has increased in the past decades. Among them Pseudomonas aeruginosa （P. aeruginosa） is one of the leading bacteria for pneumonia that are associated with poor prognosis. However, the pathogenesis of P. aeruginosa pneumonia in immunosupressed patients is not understood completely. Previous reports showed keratinocyte growth factor （KGF） is associated with lung injury in immunocompetent hosts. In this study, we investigated the different reactions of lung injury, lung pathology and KGF expressions in P aeruginosa pneumonia between immunosuppressed and immunocompetent rats. Methods Immunosuppression of male rats was induced by injecting immunosuppressive subcutaneously. Pneumonia was established by instilling P aeruginous tracheally. The immunocompetent rats were the control group. Survival rate, lung histopathology, pulmonary permeability and oedema, KGF mRNA and protein expressions in lungs of both groups were investigated. Results The survival rate of immunosuppressed group was lower than that of immunocompetent group （33.3% vs 83.3%）. After exposure to bacteria, pulmonary permeability and wet/dry ratio in immunosuppressed group were higher than those in immunocompetent group. Pulmonary congestion and haemorrhage were more intensive in immunosuppressed group compared to immunocompetent group. Apoptosis and necrosis were also observed in infected lungs of immunosuppressed rats. Although we detected KGF expressions in lungs of both groups after infection, the expressions of KGF protein and mRNA gene in immunosuppressed group were much lower than in immunocompetent group. Conclusions Compared with immunocompetent group, there was more intensive lung injury in immunosuppressed group. Severe lung injury may contribute to the poor prognosis of pneumonia. KGF expressions of pneumonia in immunosuppressed rats were less than those in immunocompetent ones.

Intestinal hormones and growth factors:Effects on the small intestine

There are various hormones and growth factors which may modify the intestinal absorption of nutrients, and which might thereby be useful in a therapeutic setting, such as in persons with short bowel syndrome. In part I, we focus first on insulin-like growth factors, epidermal and transferring growth factors, thyroid hormones and glucocorticosteroids. Part Ⅱ will detail the effects of glucagon-like peptide （GLP）-2 on intestinal absorption and adaptation, and the potential for an additive effect of GLP2 plus steroids.

Umbilical cord-derived mesenchymal stem cells regulate thymic epithelial cell development and function in Foxn 1^-/- mice

Thymic microenvironments are essential for the maturation of thymocytes, which can be anatomically compartmentalized into cortical and medullar regions. The absence of the gene encoding the transcription factor forkhead box nl （Foxnl） causes epithelial differentiation to stall in the precursor stage, resulting in the formation of an abnormal thymus. In this study, we used human umbilical cord-derived mesenchymal stem cells （UC-MSCs） to treat Foxn1^-/- mice, and then analyzed the maturation and distribution of thymic epithelial cells in the Foxn1^-/- thymic rudiment and the thymopoiesis of this newly developed rudiment. Our data showed a well-organized cortex-medulla architecture and an obvious improvement in the maturation of thymic epithelial ceils along with the appearance of UEA-1^＋MHCIIhi thymic epithelial cells in the rudiment. We further demonstrated improved thymopoiesis and the enhanced export of mature T cells with increased numbers of regulatory T cells into the peripheral blood. Furthermore, we observed that MSCs can engraft into thymic tissue and express many cytokines or proteins, particularly keratinocyte growth factor （KGF） and CD248, which are essential for thymic development. Collectively, our data identified a new mechanism for MSCs, which may provide a proper microenvironment for the reconstitution and functional maturation of the thymus in Foxn1^-/- mice. Additionally, we elicited additional insights into the therapeutic efficacy of MSCs in several autoimmune diseases.

Effect of Moxibustion on the Expressions of Protein KGF-1, KGF-2 and IL-6 in Colon of Rats with Ulcerative Colitis

Objective： To observe the effect of moxibustion on the expressions of protein keratinocyte growth factor-1 （KGF-1）, KGF-2, and interleukin-6 （IL-6） in colon of rats with ulcerative colitis （UC）, and to explore the action mechanism of moxibustion in treating UC. Methods： SD rats were randomized into a normal group, a model group, a herbs-partitioned moxibustion group, and a sulfasalazine （SASP） group. The rats in the herbs-partitioned group were treated with herbs-partitioned moxibustion at Tianshu （ST 25） and Qihai （CV 6）, and those in the SASP group were treated by intragastric administration. After interventions, HE staining and light microscope were adopted in observing the histopathological changes of rat＇s colon, and immunohistochemical methods for detecting the expressions of KGF-1, KGF-2, and IL-6 proteins in rat＇s colon. Results： Compared with the model group, the rats＇ colons in the herbs-partitioned moxibustion group and the SASP group were histopathologically improved; compared with the normal group, the expressions of KGF-1, KGF-2, and IL-6 proteins increased significantly in the model group （P〈0.05）; after intervened by herbs-partitioned moxibustion and SASP respectively, the expressions of KGF-1, KGF-2, and IL-6 proteins in rat＇s colon were decreased markedly （P〈0.05）. Conclusion： Both herbs-partitioned moxibustion and SASP can down-regulate the expressions of KGF-1, KGF-2, and IL-6 proteins in rat＇s colon, which might be one of the mechanisms of herbs-partitioned moxibustion and SASP in treating UC.

Modulation of Wnt/β-catenin signaling affects the directional differentiation of hair follicle stem cells

Aim:The differentiation of hair follicle stem cells(HFSCs)into hair follicle cells has potential clinical applications for cutaneous burns.However,the mechanisms regulating the differentiation of HFSCs into hair follicular papilla or epidermal cells are currently not clear.This study investigated the role of the Wnt/β-catenin pathway and its crosstalk with other signaling components during this differentiation process.Methods:Lithium chloride(LiCl,10 mmol/L)and keratinocyte growth factor(KGF,10μg/L)were used to induce HFSC differentiation,validated by immunofluorescence analysis.The mRNA expression ofβ-catenin,adenomatous polyposis coli,glycogen synthase kinase-3β(GSK-3β),axin,and lymphoid enhancer factor-1 after 3,5,7,and 9 days were measured to evaluate the role of the Wnt/β-catenin pathway.Results:During LiCl-induced HFSC differentiation into hair follicle cells,the Wnt/β-catenin signaling pathway was activated and the expression of GSK-3β,a vital component of the degradation compound,was inhibited.This led to increased cytoplasmicβ-catenin expression,nuclear translocation,and subsequent target gene transcription.By contrast,KGF induced the differentiation of HFSCs into epidermal cells and did not affect the expression ofβ-catenin.This data indicates that LiCl and KGF distinctly regulate the differentiation of HFSCs into hair follicle and epidermal cells,respectively.Furthermore,the Wnt/β-catenin signaling pathway is predominantly involved in hair follicle differentiation.Conclusion:these results demonstrate that LiCl can be used to differentiate HFSCs into hair follicle cells in vitro,which has important therapeutic applications for treating patients with cutaneous damage.